This project aims to develop and validate an in vitro model for studying human neuromuscular diseases. The planned experiments exploit recent advances in which human embryonic stem cells (hESCs) were differentiated into motor neurons. In preliminary investigations, hESC-derived motor neurons were cultured with murine muscle cells under conditions that promoted the formation of neuromuscular contacts. Electrophysiological analysis revealed that functional neuromuscular junctions developed within 5 days and survived for >2 weeks. The first goal of the current proposal is to establish fully human nerve-muscle cultures. Specifically, hESC-derived motor neurons will be cultured with human fetal or adult muscle cells. The initial goal will be to optimize the conditions for the formation of stable neuromuscular junctions that express markers of functional, cholinergic synapses. Concurrently, using technology in aims 2 & 3, we will assess physiological correlates of the maturation of the neuromuscular junctions. Aim 2 uses dual patch clamp analyses of neuromuscular transmission. We will characterize spontaneous and stimulus-evoked neurotransmitter release to determine whether the strength of synaptic transmission (particularly, the quantal content) increases with time in culture. If the expected strengthening o neuromuscular transmission is not observed, we will test whether changes in the culture conditions, including the addition of glia, improves the outcome. Concurrently, experiments in Aim 3 will use dynamic Ca2+ imaging to assess the reliability, kinetics and pharmacology of stimulus-dependent Ca2+ entry and Ca2+ buffering at human motor nerve terminals. As with the expected increase in synaptic strength, we anticipate that synapse maturation will lead to more-robust Ca2+ entry and efficient Ca2+ buffering at nerve terminals. Collectively, these studies will set the stage for Aim 4 in which these cultures will be transfected with a mutated form of superoxide dismutase1, which is known to cause amyotrophic lateral sclerosis (ALS) in humans. We will investigate whether the expression of this mutant enzyme affects neuromuscular function using the assays in Aims 1-3.This final set of experiments will clarify whether these cultures might be valuable assets in determining the underlying cause and possible treatment of ALS. Success of this project should lead to wider use of this model system to assess neuromuscular disease mechanisms and therapies. PUBLIC HEALTH RELEVANCE: Skeletal motor neurons control voluntary movements in humans, and in the neurodegenerative disorders, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) and spinal muscular atrophy (SMA) there is a death of skeletal motor neurons. Progress in treating these disorders has been hampered by a lack of suitable models to test prospective therapies. In this proposal, we use stem cell-derived human motor neurons to develop a model for studying human neuromuscular diseases that will set the stage for future efforts to investigate the basis of these neuromuscular disorders as well as possible therapeutic interventions.